Introduction:The aim of this study was to evaluate the effect of titanium dioxide nanoparticles (TiO2NP) addition on Endoseal MTA intra-tubular penetration after cleaning and shaping of root canal with different irrigation protocols using the confocal laser scanning microscopy (CLSM).Methods: 56 human mandibular premolars were used. The access cavities were opened. All teeth were instrumented with a crown-down technique, using a set of ProTaper rotary instruments to achieve a size #40 apical preparation. After biomechanical preparation, teeth were divided equally into 2 groups (n=28) G1 (Endoseal MTA without TiO2NP) and G2 (Endoseal MTA with 1 wt% of TiO2NP). Each group was further subdivided equally into 2 sub-groups (n=14): sub-group N (5.25% NaOCL) and sub-group T (Green tea). Each irrigant was further subdivided equally into 2 sub-groups (n=7): sub-group CI (conventional needle irrigation) and sub-group PUI (Passive ultrasonic irrigation). Sealers were mixed with the Rhodamine B fluorescent dye for analysis of sealer penetration under the CLSM at 10x magnification and obturation was carried out in lateral condensation technique. From each root 3 (1 mm) slices were obtained representing the coronal, middle and apical thirds. Results were tabulated and statistically analyzed. Results:The results showed that NaOCl groups had a significantly higher value of sealer intratubular penetration than green tea groups in both types of sealer. PUI groups had also a significantly higher value of sealer intra-tubular penetration than CI groups in both types of sealer. The results also showed that Endoseal MTA sealer without TiO2NP had a higher value of intra-tubular penetration than Endoseal MTA sealer with TiO2NP yet the difference was not statistically significant. Conclusion:Both of NaOCl and PUI permit a significantly greater sealer intra-tubular penetration compared to green tea and CI. Endoseal MTA exhibited greater intra-tubular penetration compared to Endoseal MTA with TiO2NP.